Antibacterial therapy with bacteriophage physico-chemically altered to delay inactivation by the host defense system

ABSTRACT

The present invention is directed to bacteriophage therapy, using methods which enable the bacteriophage to delay inactivation by any and all parts of the host defense system (HDS) against foreign objects. The HDS normally reduces the number of bacteriophage in an animal, which decreases the efficiency of the bacteriophage in killing the host bacteria present during an infection. Disclosed is a method of producing bacteriophage modified for anti-HDS purposes by physico-chemical alteration of the bacteriophage surface proteins, so that the altered bacteriophage remain active in the body for longer periods of time than the unmodified bacteriophage.

FIELD OF THE INVENTION

[0001] The present invention relates to a method of delaying theinactivation of bacteriophages by an animal's host defense system (HDS).One method of delaying inactivation is the use of novel bacteriophageswhose genomes have been modified. The modification of bacteriophagegenomes for the purpose of delaying inactivation is described in U.S.patent application Ser. No. ______ entitled “Antibacterial Therapy withBacteriophage Genotypically Modified to Delay Inactivation by the HostDefense system”, filed on ______, 1994, the disclosure of which isherein incorporated by reference into the present specification. Thepresent invention is directed to making a phenotypic change by attachinga polymer to phage surface proteins (i.e. physico-chemically alteringthe bacteriophage). Such polymers block or mask the phage antigenicsites from interactions with the HDS. This masking enables the alteredbacteriophage to remain in the circulation and in the tissues longerthan the unmodified phage. Thus, the altered bacteriophage is moreeffective at treating (or assisting in the treatment of) a bacterialinfection, in a human or other animal.

[0002] The present invention is also directed to specific methods ofusing the physico-chemically altered bacteriophage (whether wild-type orgenomically-modified) for treating infectious bacterial diseases. Theroute of ad-ministration can be by any means including for example,delivering the altered phage by aerosol to the lungs.

BACKGROUND OF THE INVENTION

[0003] In the 1920s, shortly after the discovery of bacterial viruses(bacteriophages), the medical community began to extensively pursue thetreatment of bacterial diseases with bacteriophage therapy. The idea ofusing phage as a therapy for infectious bacterial diseases was firstproposed by d'Herelle in 1918, as a logical application of thebacteriophages' known ability to invade and destroy bacteria. Althoughearly reports of bacteriophage therapy were somewhat favorable, withcontinued clinical usage it became clear that this form of therapy wasinconsistent and unpredictable in its results. Disappointment with phageas a means of therapy grew, because the great potential of these virusesto kill bacteria in vitro was not realized in vivo. This led to adecline in attempts to develop clinical usage of phage therapy, and thatdecline accelerated once antibiotics began to be introduced in the 1940sand 50s. From the 1960s to the present, some researchers who adoptedcertain bacteriophages as a laboratory tool and founded the field ofmolecular biology have speculated as to why phage therapy failed.

[0004] Despite the general failure of phage as therapy, isolated groupsof physicians have continued to try to use these agents to treatinfectious diseases. Many of these efforts have been concentrated inRussia and India, where the high costs of and lack of availability ofantibiotics continues to stimulate a search for alternative therapies.See for example Vopovazova et al. “Effectiveness of KlebsiellaRneumoniae Bacteriophage in the Treatment of Experimental KlebsiellaInfection”, Zhurnal Mikrobiologii. Epidemiologii Immunobiologii. pp. 5-8(April, 1991); and Voaovazova et al., “Immunological Properties andTherapeutic Effectiveness of Preparations of Klebsiella Bacteriophages”,Zhurnal Mikrobiologii. Epidemiolopii Immunobiologii. pp. 30-33 (March,1992)]. These articles are similar to most of the studies of phagetherapy, including the first reports by d'Herelle, in that they lackmany of the controls required by researchers who investigateanti-infectious therapies. In addition, these studies often have littleor no quantification of clinical results. For example, in the second ofthe two Russian articles cited above, the Results section concerningKlebsiella phage therapy states that “Its use was effective in . . .ozena (38 patients), suppuration of the nasal sinus (5 patients) and ofthe middle ear (4 patients) . . . In all cases a positive clinicaleffect was achieved without side effects from the administration of thepreparation”. Unfortunately, there were no placebo controls orantibiotic controls, and no criteria were given for “improvement”.

[0005] Another clinical use of phage that was developed in the 1950s andis currently still employed albeit to a limited extent, is the use ofphage lysate, specifically staphphage lysate (SPL). The researchers inthis field claim that a nonspecific, cell-mediated immune response tostaph endotoxin is an integral and essential part of the claimedefficacy of the SPL. [See, eg., Esber et al., J. Immunopharmacol., Vol.3, No. 1, pp. 79-92 (1981); Aoki et al., Augmenting Agents in CancerTherapy (Raven, New York), pp. 101-112 (1981); and Mudd et al., Ann. NYAcad. Sci., Vol. 236, pp. 244-251 (1974).] In this treatment, it seemsthat the purpose of using the phage is to lyse the bacteria specificallyto obtain bacterial antigens, in a manner that those authors findpreferential to lysing by sonication or other physical/chemical means.Here again, some difficulties arise in assessing these reports in theliterature, because, in general, there are no placebo controls and nostandard antibiotic controls against which to measure the reportedefficacy of the SPL. More significantly, there is no suggestion in thesearticles to use phage per se in the treatment of bacterial diseases.Moreover, the articles do not suggest that phage should be modified inany manner that would delay the capture/sequestration of phage by thehost defense system.

[0006] Since many patients will recover spontaneously from infections,studies must have carefully designed controls and explicit criteria toconfirm that a new agent is effective. The lack of quantification and ofcontrols in most of the phage reports from d'Herelle on makes itdifficult if not impossible to determine if the phage therapies have hadany beneficial effect.

[0007] As there are numerous reports of attempts at phage therapy, onewould assume that had it been effective, it would have flourished in theperiod before antibiotics were introduced. But phage therapy has beenvirtually abandoned, except for the isolated pockets mentioned above.

[0008] As noted above, some of the founders of molecular biology whopioneered the use of specific phages to investigate the molecular basisof life processes have speculated as to why phage therapy was noteffective. For example, G. Stent in his book Molecular Biology ofBacterial Viruses, WH Freeman & Co. (1963) pp. 8-9, stated thefollowing:

[0009] “Just why bacteriophages, so virulent in their antibiotic actionin vitro, proved to be so impotent in vivo, has never been adequatelyexplained. Possibly the immediate antibody response of the patientagainst the phage protein upon hypodermic injection, the sensitivity ofthe phage to inactivation by gastric juices upon oral administration,and the facility with which bacteria acquire immunity or sportresistance against phage, all militated against the success of phagetherapy.”

[0010] In 1973, Dr. Carl Merril discovered along with his co-workersthat phage lambda, administered by various routes (per os, IV, IM, IP)to germ-free, non-immune mice, was cleared out of the blood stream veryrapidly by the organs of the reticulo-endothelial system, such as thespleen, liver and bone marrow. [See Geier, Trigg and Merril, “Fate ofBacteriophage Lambda in Non-Immune, Germ-Free Mice”, Nature. 246, pp.221-222 (1973).] These observations led Dr. Merril and his co-workers tosuggest (in that same Nature article cited above) overcoming the problemby flooding the body with colloidal particles, so that thereticulo-endothelial system would be so overwhelmed engulfing theparticles that the phage might escape capture. Dr. Merril and hisco-workers did not pursue that approach at the time as there was verylittle demand for an alternative antibacterial treatment such as phagetherapy in the 1970s, given the numerous and efficacious antibioticsavailable.

[0011] Subsequently, however, numerous bacterial pathogens of greatimportance to mankind have become multidrug resistant (MDR), and theseMDR strains have spread rapidly around the world. As a result, hundredsof thousands of people now die each year from infections that could havebeen successfully treated by antibiotics just 4-5 years ago. [See e.g.C. Kunin, “Resistance to Antimicrobial Drugs - A Worldwide Calamity”,Annals of Internal Medicine, 1993;118:557-561; and H. Neu, “The Crisisin Antibiotic Resistance”, Science 257, 21 Aug 1992, pp. 1064-73.] Inthe case of MDR tuberculosis, e.g., immunocompromised as well asnon-immunocompromised patients in our era are dying within the firstmonth or so after the onset of symptoms, despite the use of as many as11 different antibiotics.

[0012] Medical authorities have described multidrug resistance not justfor TB, but for a wide variety of other infections as well. Someinfectious disease experts have termed this situation a “global crisis”.A search is underway for alternative modes and novel mechanisms fortreating these MDR bacterial infections.

[0013] Bacteriophage therapy offers one possible alternative treatment.Learning from the failure of bacteriophage therapy in the past, thepresent inventors have discovered effective ways to overcome the majorobstacles that were the cause of that failure.

[0014] One object of the present invention is to develop a drug deliveryvehicle wherein bacteriophages are protected by attaching to theirsurfaces a substance that can mask the phage surface antigens. Thismasking can be achieved by means such as, but not limited to, attachingsubstances in close proximity to the antigenic site, or attachingsubstances directly into the antigenic site, in either case, therebyblocking the host defense system's components from making contact withthe antigenic site. The purpose of masking the antigenic site is toenable a bacteriophage to delay being inactivated by the host defensesystem.

[0015] Substances which can be used to mask phage surface antigensinclude a variety of polymers, both synthetic and natural, including butnot limited to: polyethers, such as polyethylene glycol, polypropyleneglycol, polypropylene oxide, polyvinylpyrrolidone, polyvinyl alcohol,polybutanediol, polysaccharides, hyaluronic acid, collagen, albumin,dextran, carboxymethylcellulose, and poly-D,L-amino acids.

[0016] Polyethylene glycol (PEG) is a well established immune systemmodifier already in clinical use, one of its major properties being itsability to protect the antigenic sites of proteins from interaction withthe immune system.

[0017] PEG adducts are known in the art to prolong the circulating lifeof proteins that interact with the HDS. [See e.g. Nucci, M. L. et al.,The Therapeutic Value of Poly(ethylene glycol)—Modified Proteins,Advanced Drug Delivery Reviews, 6, 1991, 133-151]. In this way, a shellof PEG molecules around one or more of the antigenic proteins of thephage will sterically hinder those proteins from interacting withcomplement, with immune cells, or with any other aspect of the HDS.

[0018] One example of the use of PEG to sterically prevent theinteraction of the HDS with the antigens of a protein, is the drugADAGENT™ (pegademase bovine). This PEG-modified protein is currentlymarketed for the treatment of severe combined immunodeficiency disease(SCID), a disease which is associated with adenosine deaminasedeficiency. PEGylation of the enzyme slows its degradation and therebyrenders it efficacious as a therapeutic. [See e.g. Hershfield, M. et al,Treatment of Adenosine Deaminase Deficiency with PolyethyleneGlycol-Modified Adenosine Deaminase, New England Journal of Medicine,316:589-596 Mar. 5, 1987.] One of the derivatives of PEG that isreported to have great stability, as well as high affinity andselectivity as a linker to antigens that are to be masked, ismonomethoxypoly(ethylene glycol) (mPEG).

[0019] There are a number of methods known in the art to activatepolymers so that they will bind with the target protein. The reagents tobe used in the present invention, and known in the art for theactivation of polymers for binding, include: trichloro-s-triazine(cyanuric chloride); carbonyldiimidazole; succinic anhydride; andsuccinimidyl carbonate. Succinimidyl carbonate is preferred for use inthe present invention. The adduct targets on the protein include (butare not limited to): specific amino acid groups, sulfhydryl groups, andor other applicable moieties of the phage surface antigens that are tobe masked.

[0020] The physico-chemical alteration of bacteriophage in the presentinvention allows a delay in their inactivation by the HDS, so that thephage are no longer prevented by the HDS from reaching and killing thetarget bacteria. The masking of the phage antigenic sites also decreasesthe tendency for the human or animal recipient of the phage therapy toform antibodies against the phage. As a result, the phage therapyremains useful for longer periods of time, and/or for more courses oftreatment.

[0021] In the present invention, the adduct of the polymer with phagesurface proteins is custom designed by methods known in the art, e.g.by: 1) varying the molecular weight of the polymer, 2) altering thereaction variables, for example: the concentrations of the reagents(such as the molecule used to activate the PEG reaction); the timecourse of the reaction (this changes the percentage of the amino acidgroups of the phage surface antigen that become modified); thetemperature; the pH; etc., 3) altering the type of PEG activator beingused; and/or 4) altering the PEG derivative chosen for the reaction—oneexample among many being the bifunctional analog of SC-PEG known aspoly(ethylene glycol)-bis-N-succinimidyl carbonate (“BSC-PEG”). Thesealterations provide a variety of physico-chemically alteredbacteriophages, from which the ones demonstrating the best ability todelay inactivation by the HDS can be selected.

[0022] Given that the chemical substances linked to the bacteriophagescannot be genetically transmitted to progeny, it follows that thedaughter phages will have no protection (by physico-chemical alteration)from the HDS. Nevertheless for each physico-chemically alteredbacteriophage that does succeed in infecting a bacterium, on average afew hundred daughter phages are released within about a half hour (theactual number released and the time to burst depend on factors includingthe strain of bacteria and the strain of phage). Many of these daughterphage then have the opportunity to infect nearby bacteria, before theHDS has time to inactivate them. Therefore the rate at which the phageare multiplying is greater than the rate at which they are being takenout (by phagocytosis, complement fixation, or any other aspect of thehost defense system), resulting in exponential growth in the number ofphages at the site of an infection. Thus the physico-chemically alteredphage of the present invention establish a “beachhead”, wherein thesucceeding “waves” of bacteriophage (the first “wave” being the parentgeneration of physico-chemically altered phage, and the following“waves” being the succeeding generations of unmodified daughter phages)combine to substantially eliminate the infectious bacteria.

[0023] While PEG and the other polymers listed above are examples ofsubstances which can protect proteins from interaction with the HDS,there are many other suitable substances. Such substances are known tothose skilled in the art and any of these substances can be used in thepresent invention.

[0024] Another object of the present invention is to develop a methodfor treating bacterial infectious diseases in an animal by administeringto the animal, by an appropriate route of administration, an effectiveamount of the physico-chemically altered bacteriophage.

SUMMARY OF THE INVENTION

[0025] In the present invention, bacteriophages are physico-chemicallyaltered to produce bacteriophages capable of delaying inactivation byany component of an animal's host defense system (HDS) against foreignbodies. The physico-chemical alteration can be by attachment of suitablepolymers, or of any other suitable substance. The term “physico-chemicalalteration” applies to all polymers that can physico-chemically modify aphage in such a way as to delay its inactivation by the HDS. Thesephage, referred to as “anti-HDS physico-chemically altered phage”, areable to survive for longer periods of time in the circulation and in thetissues of an animal than the unmodified phage, thereby prolongingviability and making the phage more efficient at reaching and invadingthe bacteria at the site of an infection.

[0026] One embodiment of the present invention is the administration ofthe physico-chemically altered phage via aerosol to the lungs. This modeof administration is indicated in bacterial infections of the lungs,such as tuberculosis.

[0027] The administration of an anti-HDS modified phage will enable theanimal recipient to efficaciously fight an infection with thecorresponding bacterial pathogen. The phage therapy of this inventionwill therefore be useful either as an adjunct to standard anti-infectivetherapies, or as a stand-alone therapy.

[0028] The phages of the present invention can be administered by anyroute, such as oral, pulmonary (by aerosol or by other respiratorydevice for respiratory tract infections), nasal, IV, IP, per vagina, perrectum, intra-ocular, by lumbar puncture, intrathecal, and by burr holeor craniotomy if need be for direct insertion onto the meninges (e.g. ina heavily thickened and rapidly fatal tuberculous meningitis).

DETAILED DESCRIPTION OF THE INVENTION

[0029] One of the major obstacles to bacteriophage therapy is the factthat when phages are administered to animals, they are rapidlyeliminated by the animal's HDS. That suggests that the phages are notviable in the animal's circulation or tissues for a long enough time toreach the site of infection and invade the bacteria. Thus, the object ofthe present invention is to physico-chemically alter the bacteriophages,so as to delay inactivation by the HDS. This will prolong phageviability in the body.

[0030] The attachment of a polymer to the bacteriophage increases itscirculating life and reduces its immunogenicity and antigenicity. Oneexample of such a polymer is PEG (polyethylene glycol). PEG is a linear,uncharged, flexible polymer available in a variety of molecular weights.In the present invention, the PEG strands are attached to the phage andsterically block the antigenic site from antibody attachment. Prior toPEGylating the bacteriophage, the tail proteins of the phages areprotected from being PEGylated so as to protect those tail proteins fromthe steric hindrance that PEGylation would otherwise induce. The tailportion of the phage must be protected from PEGylation to ensure that itwill adhere to the host bacteria so that it can inject its DNA into thebacteria. The steric hindrance caused by polymers (such as polyethyleneglycol) is stated in the art as being the likely mechanism by which saidpolymers are able to protect protein antigenic sites from interactingwith the immune system. [See e.g. Davis, F. F. et al. (1991) Reductionof immunogenicity and extension of circulation half-life of peptides andproteins. In: V. H. L. Lee (Ed.), Peptide and Protein Drug Delivery,Marcel Dekker, N.Y., pp. 831-864.] However, the proteins ofbacteriophage tails are in general the specific means by whichbacteriophage adhere to and subsequently infect the host bacteria.Therefore any steric hindrance of the phage tail proteins themselves,which might be induced by the attachment of PEG or other polymers in thepresent invention, is to be avoided.

[0031] PEG has been approved by the Food and Drug Administration (FDA)as a vehicle or base for a number of pharmaceutical preparations, andhas a low order of toxicity when administered orally and parenterally.

[0032] The term “host defense system” as used herein refers to all ofthe various structures and functions that help an animal to eliminateforeign bodies. These defenses include but are not limited to the formedcells of the immune system and the humoral components of the immunesystem, those humoral components including such substances ascomplement, lysozymes and beta-lysin. In addition, the organs of whathas often been referred to as the “reticulo-endothelial system” (spleen,liver, bone marrow, lymph glands, etc.) also serve as part of the hostdefense system, In addition to all the phenomena cited just above whichtake place within this “reticulo-endothelial system”, there has alsobeen described a sequestering action wherein foreign materials(specifically including bacteriophage) are captured non-phagocyticallyand non-destructively in the spleen by what is known as theSchweigger-Seidel capillary sheaths [See e.g. Nagy, Z., Horrath, E., andUrban, Z., Nature New Biology, 242: p. 241 (1973).]

[0033] The phrase “substantially eliminate” as used regarding thepresent invention, indicates that the bacteria are reduced to a numberwhich can then be completely (or at least sufficiently) eliminated bythe animal's defense system, or by using conventional antibacterialtherapies.

[0034] Enabling bacteriophages to delay inactivation by those hostdefense systems—whichever components of it may or may not be employed inany given case—would be likely to result in an increased in vivo killingof bacterial pathogens that are in the host range for thosebacteriophages.

[0035] As used herein, the term “anti-HDS physico-chemically alteredphage” is intended to cover not only those phages that have beenmodified by the attachment of various PEG derivatives, but also phagesthat have been modified by any of the numerous additional polymers knownin the art. Such “physico-chemically altered” phages may be wild-type,or alternatively may have been previously modified by other procedures,such as genomic alterations (made by serial passage or by geneticengineering techniques). Further, a “physico-chemically altered” phage,as used herein, has a half-life within the animal that is at least 15%greater than the half-life of the original unmodified phage from whichit was derived. Half-life refers to the point in time when, out of aninitial IV dose (e.g. 1×10¹²) of a given phage, half (1×10⁶) of themstill remain in circulation, as determined by serial pfu experiments(“pfu” meaning plaque forming units, a convenient measure of how manyphage are present in a given sample being assayed). A 15% longerhalf-life indicates a successful delay of inactivation by the HDS. Inthe case where a polymer is attached to a phage that is already able todelay inactivation by virtue of genotypic changes, then the additionalmodification of the present invention imparts an increase of at least anadditional 15%, above and beyond the baseline extended half-life thathas already been conferred by virtue of said genotypic changes.

[0036] Additional evidence that the HDS-evading phages do in fact remainviable for a longer period of time in the body is obtained bydemonstrating not only the longer time that they remain in thecirculation (see above), but also by the higher numbers of them thatremain in the circulation at a given point in time. This slower rate ofclearance can be demonstrated by the fact that e.g. ten minutes afterthe IV injection of 1×10¹² of the anti-HDS modified phage into a testanimal, the number of the phages still in circulation (as measured bypfu assays) is at least 10% higher than the number of the correspondingunmodified phage still in circulation in the control animal, at thatpoint in time.

[0037] The present invention can be applied across the spectrum ofbacterial diseases, through attachment of suitable polymers to wild-typephage or to genotypically modified phage, so that anti-HDS modifiedphages are developed that are specific for each of the bacterial strainsof interest. In that way, a full array of anti-HDS modified phages canbe developed for virtually all the bacterial pathogens of man, his pets,livestock and zoo animals (whether mammal, avian, or pisciculture).Anti-HDS modified phage therapy will then be available:

[0038] 1) as an adjunct to or as a replacement for those antibioticsand/or chemotherapeutic drugs that are no longer functioning in abacteriostatic or bactericidal manner due to the development ofmulti-drug resistance;

[0039] 2) as a treatment for those patients who are allergic to theantibiotics and/or chemotherapeutic drugs that would otherwise beindicated; and

[0040] 3) as a treatment that has fewer side effects than many of theantibiotics and/or chemotherapeutic drugs that would otherwise beindicated for a given infection.

[0041] The second embodiment of the present invention is the developmentof methods to treat bacterial infections in animals through phagetherapy with the anti-HDS physico-chemically altered bacteriophages.Hundreds of bacteriophages and the bacterial species they infect areknown in the art. The present invention is not limited to a specificbacteriophage or a specific bacteria. Rather, the present invention canbe utilized to develop anti-HDS physico-chemically alteredbacteriophages which can be used to treat any and all infections causedby their host bacteria.

[0042] While it is contemplated that the present invention can be usedto treat any bacterial infection in an animal, it is particularlycontemplated that the methods described herein will be very useful as atherapy (adjunctive or stand-alone) in infections caused bydrug-resistant bacteria. Experts report [See e.g. Gibbons, A.,“Exploring New Strategies to Fight Drug-Resistant Microbes, Science. 21Aug. 1993, pp. 1036-38.] that at the present time, the drug-resistantbacterial species and strains listed below represent the greatest threatto mankind:

[0043] 1. All of the clinically important members of the familyEnterobacteriaceae, most notably but not limited to the following:

[0044] a) All the clinically important strains of Escherichia, mostnotably E. coli. One among a number of possible wild-type phages againstthese particular pathogens that could be used as a starting point forthe serial passage and/or the genetic engineering of the presentinvention would be ATCC phage # 23723-B2. [Note: For purposes ofbrevity, in all the following examples of pathogens, the correspondingwild-type phage will be indicated by the following phraseology: “Exampleof corresponding phage:______”.]

[0045] b) All the clinically important strains of Klebsiella, mostnotably K. pneumoniae [Example of corresponding phage: ATCC phage #23356-B1].

[0046] c) All the clinically important strains of Shigella, most notablyS. dysenteriae [Example of corresponding phage: ATCC phage # 11456a-B1].

[0047] d) All the clinically important strains of Salmonella, includingS. abortus-equi [Example of corresponding phage: ATCC phage # 9842-B1],S. typhi [Example of corresponding phage: ATCC phage # 19937-B1], c.typhimurium [Example of corresponding phage: ATCC phage # 19585-B1], S.newport [Example of corresponding phage: ATCC phage # 27869-B1], S.paratyphi-A [Example of corresponding phage: ATCC phage # 12176-B1], S.paratyphi-B [Example of corresponding phage: ATCC phage # 19940-B1], S.Potsdam [Example of corresponding phage: ATCC phage # 25957-B2], and S.pollurum [Example of corresponding phage: ATCC phage # 19945-B1].

[0048] e) All the clinically important strains of Serratia, most notablyS. marcescens [Example of corresponding phage: ATCC phage # 14764-B1]).

[0049] f) All the clinically important strains of Yersinia, most notablyY. pestis [Example of corresponding phage: ATCC phage # 11953-B1].

[0050] g) All the clinically important strains of Enterobacter, mostnotably E. cloacae [Example of corresponding phage: ATCC phage #23355-B1].

[0051] 2. All the clinically important Enterococci, most notably E.faecalis [Example of corresponding phage: ATCC phage # 19948-B1] and E.faecium [Example of corresponding phage: ATCC phage #19953-B1].

[0052] 3. All the clinically important Haemophilus strains, most notablyH. influenzae [a corresponding phage is not available from ATCC for thispathogen, but several can be obtained from WHO or other labs that makethem available publicly].

[0053] 4. All the clinically important Mycobacteria, most notably M.tuberculosis [Example of corresponding phage: ATCC phage # 25618-B1], M.avium-intracellulare, M. bovis, and M. leprae. [Corresponding phage forthese pathogens are available commercially from WHO, via The NationalInstitute of Public Healthy & Environmental Protection, Bilthoven, TheNetherlands].

[0054] 5. Neisseria gonorrhoeae and N. meningitidis [Corresponding phagefor both can be obtained publicly from WHO or other sources].

[0055] 6. All the clinically important Pseudomonads, most notably P.aeuruginosa [Example of corresponding phage: ATCC phage # 14203-B1].

[0056] 7. All the clinically important Staphylococci, most notably S.aureus [Example of corresponding phage: ATCC phage # 27690-B1] and S.epidermidis [corresponding phage are available publicly through the WHO,via the Colindale Institute in London].

[0057] 8. All the clinically important Streptococci, most notably S.pneumoniae [Corresponding phage can be obtained publicly from WHO orother sources].

[0058] 9. Vibrio cholera [Example of corresponding phage: ATCC phage #14100-B1].

[0059] There are additional bacterial pathogens too numerous to mentionthat, while not currently in a state of antibiotic-resistance crisis,nevertheless make excellent candidates for treatment with anti-HDSphysico-chemically altered bacteriophages that are able to delayinactivation by the HDS in accordance with the present invention. Thus,all bacterial infections caused by bacteria for which there is acorresponding phage can be treated using the present invention.

[0060] Any phage strain capable of doing direct or indirect harm to abacteria is contemplated as useful in the present invention. Thus,phages that are lytic, phages that are lysogenic but can later becomelytic, and nonlytic phages that can deliver a product that will beharmful to the bacteria are all useful in the present invention.

[0061] The animals to be treated by the methods of the present inventioninclude but are not limited to man, his domestic pets, livestock,pisciculture, and the animals in zoos and aquatic parks (such as whalesand dolphins).

[0062] The anti-HDS physico-chemically altered bacteriophages of thepresent invention can be used as a stand-alone therapy or as anadjunctive therapy for the treatment of bacterial infections. Numerousantimicrobial agents (including antibiotics and chemotherapeutic agents)are known in the art which would be useful in combination with anti-HDSphysico-chemically altered bacteriophages for treating bacterialinfections. Examples of suitable antimicrobial agents and the bacterialinfections which can be treated with the specified antimicrobial agentsare listed below. However, the present invention is not limited to theantimicrobial agents listed below, as one skilled in the art couldeasily determine other antimicrobial agents useful in combination withanti-HDS physico-chemically altered bacteriophage therapy. PathogenAntimicrobial or antimicrobial group E. coli uncomplicated urinarytrimethoprim-sulfamethoxazole tract infection (abbrev. TNO-SMO), orampicillin; 1st generation cephalosporins, ciprofloxacin systemicinfection ampicillin, or a 3rd generation cephalosprorin;aminoglycosides, aztreonam, or a penicillin + a pencillinase inhibitorKiebsiella pneumoniae 1st generation cephalosporins; 3rd gener.cephalosporins, cefotaxime, moxalactam, amikacin, chloramphenicolShigella (various) ciprofloxacin; TMO-SMO, ampicillin, chloramphenicolSalmonella: S. typhi chloramphenicol; ampicillin, TMO-SMO non-typhispecies ampicillin; chloramphenicol, TMO-SMO, ciprofloxacin Yersiniapestis streptomycin; tetracycline, chlor- amphenicol Enterobactercloacae 3rd generation cephalosporins, gentamicin, or tobramycin;carbenicillin, amikacin, aztreonam, imipenem Hemophilus influenzae:meningitis chloramphenicol or 3rd generation cephalosporins; ampicillinother H. infl. infections ampicillin; TMO-SMO, cefaclor, cefuroxime,ciprofloxacin Mycobacterium tuberculosis isoniazid (INH) + rifampin orand M. avium-intracellulare rifabutin, the above given along withpyrazinamide +/or ethambutol Neisseria: N. meningitidis penicillin G;chloramphenicol, or a sulfonamide N. gonorrhoeae: penicillin-sensitivepenicillin G; spectinomycin, ceftriaxone penicillin-resistantceftriaxone; spectinomycin, cefuroxime or cefoxitin, cipro- floxacinPseudomonas aeruginosa tobramycin or gentamycin (+/− carben- icillin,aminoglycosides); amikacin, ceftazidime, aztreonam, imipenem Staphaureus non-penicillinase penicillin G; 1st generation cephalo- producingsporins, vancomycin, imipenem, erythromycin penicillinase producing apenicillinase-resisting penicillin; 1st generation cephalosporins,vanco- mycin, imipenem, erythromycin Streptococcus pneumoniae penicillinG; 1st gener. cephalo- sporins, erythromycin, chloram- phenicol Vibriocholera tetracycline; TMO-SMO

[0063] The routes of administration include but are not limited to:oral, aerosol or other device for delivery to the lungs, nasal spray,intravenous, intramuscular, intraperitoneal, intrathecal, vaginal,rectal, topical, lumbar puncture, intrathecal, and direct application tothe brain and/or meninges. Excipients which can be used as a vehicle forthe delivery of the phage will be apparent to those skilled in the art.For example, the physico-chemically altered phage could be inlyophilized form and be dissolved just prior to administration by IVinjection. The dosage of administration is contemplated to be in therange of about 10⁶ to about 10¹³ pfu/per kg/per day, and preferablyabout 10¹² pfu/per kg/per day. The phage are administered untilsufficient elimination of the pathogenic bacteria is achieved.

[0064] With respect to the aerosol administration to the lungs, theanti-HDS physico-chemically altered phages are incorporated into anaerosol formulation specifically designed for administration to thelungs by inhalation. Many such aerosols are known in the art, and thepresent invention is not limited to any particular formulation.

[0065] An example of such an aerosol is the Proventil™ inhalermanufactured by Schering-Plough, the propellant of which containstrichloromonofluoromethane, dichlorodifluoromethane and oleic acid. Theconcentrations of the propellant ingredients and emulsifiers areadjusted if necessary based on the phage being used in the treatment.The number of phages to be administered per aerosol treatment will be inthe range of 10⁶ to 10¹³ pfu, and preferably 10¹² pfu.

[0066] For wild-type phage that have not been genotypically modified todelay inactivation by the HDS, physico-chemical alteration will providea means for such phage to delay that inactivation. For phage that havebeen genotypically modified to delay inactivation by the HDS,physico-chemical alteration will provide an additional and synergisticmeasure of protection against such HDS inactivation. Furthermore, forboth the genotypically-modified and the wild-type phage,physico-chemical alteration will shield the phages from immune cells andcomplement, minimizing the rate of antibody formation against the phageantigens.

[0067] In summary, the use of physico-chemically altered bacteriophageto delay inactivation by the HDS provides the following advantages: 1)it enables the use of smaller whole body dosages of phage than would berequired with non-modified phage; 2) because of the overall lower wholebody dosages required and the fact that the phage are sheltered from theHDS, there is a delay and a minimization of antibody-antigeninteractions, so that 3) the physico-chemical alteration prolongs thenumber of weeks or months that phage therapy may remain effective in agiven animal, and increases the number of courses of phage therapy thatmay usefully be given over time.

[0068] The foregoing embodiments of the present invention are furtherdescribed in the following Examples. However, the present invention isnot limited by the Examples, and variations will be apparent to thoseskilled in the art without departing from the scope of the presentinvention. In particular, any bacteria and phage known to infect saidbacteria can be substituted in the experiments of the followingexamples.

EXAMPLES Example 1 PEGvlating Bacteriophage to Block their AntigenicSites from Interaction with the Host Defense System

[0069] Part 1. Protecting the tail portion of the phage from PEGylation

[0070] Tails of a lambda coliphage are isolated by sonication andcentrifugation. The tail particles resulting from sonication areinjected into rabbits to raise antibodies to the tail proteins,following procedures known in the art. The anti-tail antibodies are thenimmobilized on a solid support column using CNBR-activated sepharose 4B(Pharmacia, Piscataway, N.J.). An excess number of whole, intact lambdaphage are incubated with the column-immobilized anti-tail antibodies, toallow the formation of complexes between the tail antigens of the intactphage and the anti-tail antibodies immobilized on the support column.The formation of these complexes immobilizes the intact phage by theirtail portions only, leaving all non-tail proteins exposed to thePEGylation reagents. Free phage that have not been immobilized areeluted with normal saline, and discarded.

[0071] Part 2. PEGylation of the phage

Step 1. Preparation of the PEG Derivative

[0072] The PEG derivative used is monomethoxypoly(ethylene glycol),abbreviated “mPEG”. The mPEG is activated with succinimidyl carbonate,by methods known in the art [See e.g. Zalipsky et. al., Use offunctionalized polyethylene glycols for modification of poly-peptides,in press.] In this method, mPEG, of molecular weight 5000 (UnionCarbide, 60 g, 12 mmol), dried by azeotropic removal of toluene, isdissolved in toluene/dichloromethane (3:1, 200 ml) and treated under awell-ventilated hood with a toluene solution of phosgene (30 ml, 57mmol) overnight. The solution is evaporated to dryness and the remainderof the phosgene is removed under vacuum, under the hood. The residue isredissolved in toluene/dichloromethane (2:1, 150 ml) and treated withsolid N-hydroxysuccinimide (2.1 g, 18 mmol) followed by triethylamine(1.7 ml, 12 mmol). After 3 hours the solution is filtered and evaporatedto dryness. The residue is dissolved in warm (50° C.) ethyl acetate (600ml) and trace insolubles are filtered out. The residue is cooled tofacilitate precipitation of the polymer. The product is collected byfiltration and then recrystallized once more from ethyl acetate. Theproduct is dried in vacuo over P₂O₅. The resulting product ismethoxypoly(ethylene glycol)-N-succinimidyl carbonate (“SC-PEG”).

Step 2. Altering the Phage Proteins using SC-PEG

[0073] SC-PEG is mixed into a quantity of 0.1 M sodium phosphate (pH7.8) sufficient to completely fill the column of immobilized phages andto achieve a concentration of 0.033 mmol SC-PEG per ml of sodiumphosphate. The column is filled completely with the mixture, addingsodium hydroxide (0.5 N) as needed to maintain pH 7.8 for the durationof the reaction, which is terminated at 30 min. The excess of freeSC-PEG is removed by diafiltration using 50 mM phosphate bufferedsaline. The altered phage are separated from the column-bound anti-tailantibodies by eluting with glycine buffer at pH 2.6, then immediatelyneutralizing to pH 7.5 with TRIS base.

[0074] Part 3. Determining the Extent of PEGylation of the PhageProteins

[0075] The number of amino groups of the phage proteins that havereacted with SC-PEG is measured using the trinitrobenzenesulfonate(TNBS) assay, [See e.g. Habeeb, A. Anal. Biochem. 1966, 14, 328]

Example 2 Demonstration that PEGvlated phage remain lytic in vitro

[0076] A 100 cc broth containing the host strain of coliphage (in aconcentration of 1×10¹² bacteria/cc) is inoculated with 1×10¹² PEGylatedphage suspended in 1 cc of sterile normal saline. A control broth of thesame bacteria is inoculated in the same way, with the unmodifiedcoliphage. The determination that both the modified and the unmodifiedphage lyse their respective broths in roughly equal periods of time isshown by:

[0077] a) roughly equal turbidimetric measures in both broths;

[0078] b) sterility in both broths, evidenced by no bacterial growth onagar, from plating out of both lysed broths; and

[0079] c) pfu experiments showing that both cleared broths haveconcentrations of daughter phage at least 1000 times higher than theconcentrations of phage in the broths at time zero.

Example 3 Demonstration that PEGvlated Phage take Longer to beInactivated by the HDS as Compared to Unmodified Phage

[0080] Two groups of mice are injected with phage as specified below:

[0081] Group 1: The experimental group receives an IV injectionconsisting of 1×10¹² of the PEGylated phage, suspended in 0.5 cc ofsterile normal saline.

[0082] Group 2: The control group receives an IV injection consisting of1×10¹² of the unmodified phage from which the modified phage werederived, suspended in 0.5 cc of sterile normal saline.

[0083] Both groups of mice are bled at regular intervals, and the bloodsamples are assayed for phage content (by pfu assays) to determine thefollowing:

[0084] 1) Assays for half-life of the two phages: For each group ofmice, the point in time is noted at which there remains in circulationonly half (i.e., 1×10⁶) the amount of phage administered at the outset.

[0085] 2) Assays for absolute numbers: For each group of mice, a sampleof blood is taken at precisely 1 hour after administration of the phage.

Example 4 Demonstration that PEGvlated Phage Remain Lytic in vivo, andhave a better Ability than Unmodified Phage to Prevent a LethalInfection

[0086] Part 1. Peritonitis Model

[0087] An LD₅₀ dosage of E. coli is administered intraperitonally (IP)to laboratory mice. The strain of E. coli used is one known to be lysedby the coliphage strain that has been PEGylated. The treatment modalityis administered precisely 20 minutes after the bacteria are injected,but before the onset of symptoms. The treatment modalities consist ofthe following:

[0088] Group 1: The experimental group receives an IP injectionconsisting of 1×10¹² of the PEGylated lambda coliphage, suspended in 2cc of sterile normal saline.

[0089] Group 2: A first control group receives an IP injectionconsisting of 1×10¹² of the unmodified phage, suspended in 2 cc ofnormal sterile saline.

[0090] Group 3: A second control group receives an IP injection ofsterile normal saline.

[0091] The following criteria are measured to determine theeffectiveness of the PEGylated phage.

[0092] (1) Survival of the animals: Survival rates of the mice receivingthe PEGylated phage are compared to the survival rates of the micereceiving the unmodified phage.

[0093] (2) Bacterial counts: Samples of peritoneal fluid are withdrawnevery ½hour from the three groups of infected mice, and are streaked onculture dishes. The rate of decrease in E. coli colony counts in thethree groups is compared.

[0094] (3) Phage counts: Samples of peritoneal fluid from the differentgroups of mice are compared in plaque forming unit experiments.

[0095] Part 2. Bacteremia Model

[0096] An LD₅₀ dosage of E. coli is administered intravenously (IV) tolaboratory mice, where the strain of E. coli used is known to be lysedby the coliphage strain that was PEGylated. The treatment modality (seebelow) is administered precisely 20 minutes after the bacteria areinjected, but before the onset of symptoms. All groups are bled on anhourly basis. The treatment modalities consist of the following:

[0097] Group 1: The experimental group receives an IV injectionconsisting of 1×10¹² of the PEGylated lambda coliphage, suspended in 0.5cc of sterile normal saline.

[0098] Group 2: A first control group receives an IV injectionconsisting of 1×10¹² of the unmodified phage, suspended in 0.5 cc ofsterile normal saline.

[0099] Group 3: A second control group receives an IV injection of 0.5cc of sterile normal saline.

[0100] The following criteria are measured to determine theeffectiveness of the PEGylated phage.

[0101] (1) Survival of the animals: Survival rates of the mice receivingthe PEGylated phage are compared to the survival rates of the micereceiving the unmodified phage.

[0102] (2) Bacterial counts: The serial blood samples are streaked onculture dishes. The rate of decrease in E. coli colony counts in thethree groups is compared.

[0103] (3) Phage counts: The serial blood samples are tested in plaqueforming unit experiments. The number of plaque forming units in thethree groups is compared.

We claim:
 1. A method for treating an infectious disease caused by abacteria, in an animal, comprising: administering to an animal in needof such treatment, a lytic or non-lytic physico-chemically alteredbacteriophage that is specific for said bacteria, in a dosage effectiveto substantially eliminate the bacteria, wherein said physico-chemicallyaltered bacteriophage has a delayed inactivation by an animal's hostdefense system (HDS).
 2. The method according to claim 1 , wherein saidbacteria is a drug resistant bacteria.
 3. The method according to claim1 , wherein said animal is not a mammal.
 4. The method according toclaim 1 , wherein said animal is a mammal.
 5. The method according toclaim 4 , wherein said mammal is a human.
 6. The method according toclaim 1 , wherein said physico-chemically altered bacteriophage isPEGylated.
 7. The method according to claim 1 , wherein saidphysico-chemically altered bacteriophage has at least a 15% longerhalf-life than a corresponding wild-type phage.
 8. The method accordingto claim 1 , wherein the bacteria is selected from the group consistingof Mycobacteria, Staphylococci, Vibrio, Enterobacter, Enterococci,Escherichia, Haemophilus, Neisseria, Pseudomonas, Shigella, Serratia,Salmonella and Streptococci, and the bacteriophage can effectively lysethe bacteria.
 9. The method according to claim 8 , wherein the bacteriais selected from the group consisting of M. tuberculosis, M.avium-intracellulare and M. bovis.
 10. The method according to claim 1 ,wherein the bacteriophage is administered by way of an aerosol to ananimal's lungs.
 11. The method according to claim 1 , wherein thebacteriophage is administered at a dosage of about 10⁶ to about 10¹³pfu/kg/day.
 12. The method according to claim 11 , wherein thebacteriophage is administered at a dosage of about 10¹² pfu/kg/day. 13.The method according to claim 1 , wherein said bacteriophage isgenetically modified to evade the HDS.
 14. A physico-chemically alteredbacteriophage which is able to delay inactivation by an animal's hostdefense system.
 15. The bacteriophage according to claim 14 , whereinsaid bacteriophage has at least a 15% longer half-life than acorresponding wild-type phage.
 16. The bacteriophage according to claim14 , wherein said phage is specific for bacterial families selected fromthe group consisting of Escherichia, Klebsiella, Shigella, Salmonella,Serratia, Yersinia, Enterobacter, Enterococci, Haemophilus,Mycobacteria, Neisseria, Pseudomonas, Staphylococci, Streptococci andVibrio.
 17. The bacteriophage according to claim 14 , wherein saidbacteriophage is PEGylated.
 18. A method of obtaining aphysico-chemically altered bacteriophage that is able to delayinactivation by an animal's host defense system against foreign bodies,comprising the steps of: (a) protecting tail proteins on abacteriophage, and (b) then binding a polymer to any unprotectedproteins on said bacteriophage.
 19. The method according to claim 18 ,wherein said polymer is polyethylene glycol (PEG).
 20. A method fortreating an infectious disease caused by a bacteria, comprisingadministering to an animal in need of such treatment an antibioticand/or a chemotherapeutic agent, in combination with aphysico-chemically altered bacteriophage specific for said bacteria, ina dosage effective to substantially eliminate the bacteria, wherein saidphysico-chemically altered bacteriophage is able to delay inactivationby the animal's host defense system.
 21. The method according to claim20 , wherein said physico-chemically altered bacteriophage is PEGylated.22. A pharmaceutical composition comprising a physico-chemically alteredbacteriophage which is able to delay inactivation by an animal's hostdefense system, in combination with a pharmaceutically acceptablecarrier.
 23. The pharmaceutical composition according to claim 22 ,wherein said physico-chemically altered bacteriophage is PEGylated. 24.The pharmaceutical composition according to claim 22 , wherein saidcomposition is an aerosol formulation for administration to an animal'slungs.
 25. The pharmaceutical composition according to claim 22 ,wherein said bacteriophage is in lyophilized form.